This invention relates to an improved liquid chromatography absorbance detector and particularly to one that provides cancellation of the effects of refractive index gradients and other distortion sources within a sample test cell.
Liquid chromatography is a common analytical procedure used primarily for the separation and detection of substances by differential, rates of migration as the substances are equilibrated between a stationary finely divided medium contained in a narrow column, a non-volatile solvent which is pumped through the column. After the compounds have been separated by passage through the column, they must be detected and quantified. The detection/quantification process most commonly used is measurement of the absorption measurement of the molecules in the ulraviolet/visible region of the spectrum. In general, the detection process employs broad spectrum or monochromatic light which is passed through a test cell containing a liquid sample and the absorbance of selected frequencies of light is sensed by an absorbance detector and evaluated. The absorbance detector converts the incident radiation to a corresponding electrical signal which provides a measure of the absorption characteristics of the substance being characterized.
Although presently available liquid chromatography devices operate satisfactorily, there is a continuing need to improve their measurement accuracy by reducing noise which limits sensitivity. As light is transmitted through the test cell, a portion of the total energy of the light is absorbed which causes heating of the substances being evaluated. Such heating frequently causes localized refractive index variations which create tiny "lenses" which refract the light traversing the cell in a non-uniform manner. In certain liquid chromatographic techniques, the solvent mixture composition is continuously changed throughout the course of the separation in order to enhance the separation process. Since the solvent components do not usually have the same refractive indices, this process will make the optical system behave as a prism of continuously varying refractive power. If the solvent flow is not uniform, then there will be fluctuations in the refraction angle. In both cases, such randomly varying refraction causes random portions of the radiation passing through the test cell to escape detection, thereby increasing noise and decreasing measurement accuracy. The refraction effect caused by index variations is not constant with respect to time, and therefore, a static compensation approach cannot cancel distortions due to this effect. In addition to the above-mentioned phenomena, other factors such as imperfections in the optical elements of the system can cause portions of the radiation to be scattered so that each portion escapes detection.
In view of the foregoing, it is an object of this invention to provide an improved absorbance detector particularly useful for use with liquid chromatography devices. This detector compensates for the effects of distortions of the light as it passes through the test cell. In accordance with this invention, such improvements are achieved in part through the use of an approximate phase conjugator in the form of a retroreflective array. The array causes rays passing through the test cell to be redirected closely along their same paths to again pass through the test cell. Such retracing causes the rays to be distorted in a reverse sense from the original distortion, thus "undoing" the original distortion and thereby compensating for it. Thereafter, radiation which has passed through the sample is separated from incident radiation by a beam splitter. Use of the retroreflective array enables distortion compensation to occur at a rate which is much faster than the relatively slow rate of the time dependent changes within the cell. This design further causes the light rays to pass through the sample twice, thereby doubling the signal obtained from the sample.
The use of retroreflective arrays in absorbance test apparatus, in general, is previously known as exemplified by U.S. Pat. No. 3,628,872 issued to Miranda. That patent sought to eliminate the effects of the non-planar optical surfaces of a test cell such as a cylindrical test tube and employed a retroreflective array for such compensation. The chromatography device in accordance with this invention, however, differs in many fundamental respects from that described in Miranda. Miranda teaches the use of a collimated light being passed through a large sample cell in the form of a conventional test tube. In modern liquid chromatographic practices, it is necessary to limit the volume of the sample cell, usually to one microliter or less. In accordance with the present invention, light is focused within a relatively small test cell. The Miranda patent does not comprehend that the efficiency of a retroreflective array is enhanced if the light strikes it nearly perpendicularly. In accordance with this invention, the light is collimated after passing through the sample and prior to exposure to the array. A retroreflective array is not optically perfect since a portion of the light striking its surface is simply reflected such that the angle of incidence equals the angle of reflection. Such specular reflection may arise from front surface reflections and from inteference effects between beams reflected from array elements. Specular reflection constitutes a source of noise since the reflected rays do not retrace their original paths. As a means of addressing this source of error, the present invention eliminates the effects of such direct reflection by slightly tipping the array with respect to the optical axis of the system such that reflected rays escape the optical path coupled to the detectors. The Miranda patent further does not comprehend means for eliminating the effects of light that is reflected directly from the planar surfaces of the test cell. As a means of eliminating such influences, optical mechanisms are provided to prevent the detectors from being influenced by such specular reflection.
Additional benefits and advantages of the present invention will become apparent to those skilled in the art to which this invention relates from the subsequent description of the preferred embodiments and the appended claims, taken in conjunction with the accompanying drawings.